We asked whether patients with AIDS or ARC receiving long-term therapy with AZT, ddC, or ddI developed resistance to these drugs, and if so, to explore its biochemical basis. Primary HIV strains were obtained from peripheral blood mononuclear cells (PBM) from adult patients with AIDS or ARC who received an alternating regimen of AZT and ddC (AZT/ddC) for 15-41 months or who received ddI for 12-16 months. Isolates were obtained before and during antiviral therapy. All virions were isolated by coculturing patient's PBM and phytohemagglutinin (PHA)- stimulated PBM from healthy volunteers. The sensitivity of primary HIV-1 isolates to AZT, ddC or DDI was assessed by drug concentrations (uM) that brought about 50% p24 Gag negative wells (CN50) when HIV p24 Gag protein production by PHA-PBM exposed to a fixed dose of each viral isolate was determined in 10-12 days culture 4-8 replicates. We found that AZT therapy can induce AZT-resistant HIV variants as early as 2 months; and once acquired, AZT resistance may remain after switching to ddI therapy for 1 year in some patients. HIV appears to develop resistance to ddC and ddI less easily than to AZT, while these data do not provide basis for concluding that AZT/ddC or ddI are inferior, equivalent, or superior to AZT as therapy of AIDS. In addition, more research is needed to make clinical correlations between in vitro drug sensitivities of HIV and clinical outcome in HIV infection.